KC-0244

293T-human-B7H7-Cell-Line

20087

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Background of 293T-human-B7H7-Cell-Line

HHLA2, also named as B7H7, is a member of the B7 family of immune regulatory proteins, B7H7 is expressed monocytes and is up-regulated by LPS and IFN-gamma stimulation.

Specifications

Catalog Number	KC-0244
Cell Line Name	293T-human-B7H7-Cell-Line
Host Cell Line	293T
Description	Stable HEK293T clone expressing exogenous human B7H7 gene
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

293T human-B7H7 cell line was generated using a lentiviral vector expressing the human-B7H7 sequence.

Characterization

Figure 1: Characterization of human-B7H7 overexpression in the 293T human-B7H7 stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 1μg/mL puromycin)in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage.

References

Zhao, Ruihua, Jordan M Chinai, Susan Buhl, Lisa Scandiuzzi, Anjana Ray, Hyungjun Jeon, Kim C Ohaegbulam, et
al. 2013. “HHLA2 Is a Member of the B7 Family and Inhibits Human CD4 and CD8 T-Cell Function..” Proceedings
of the National Academy of Sciences of the United States of America 110 (24). National Academy of Sciences:
9879–84. doi:10.1073/pnas.1303524110.
Koirala, Pratistha, Michael E Roth, Jonathan Gill, Jordan M Chinai, Michelle R Ewart, Sajida Piperdi, David S Geller,
et al. 2016. “HHLA2, a Member of the B7 Family, Is Expressed in Human Osteosarcoma and Is Associated with
Metastases and Worse Survival.” Scientific Reports, August. Nature Publishing Group, 1–9.
doi:10.1038/srep31154.